Chronic opioid use changes the brain's state, including neuroimmune activity, impacting brain function and behavior and contributing to continued drug dependence and pathology. However, there is little work examining the progression of the cytokine response during opioid dependence and withdrawal, a period of profound autonomic and emotional upset. Our recent published and preliminary data demonstrate a significant neuroimmune response during withdrawal. These findings suggest that an abrupt cessation of opioid intake leads to an acute central nervous system (CNS) inflammatory response in the central nucleus of the amygdala (CeA), a brain region neuroanatomically associated with affective and cardiorespiratory regulation. It is now reasonable to postulate a chain of proinflammatory responses in a network involving vascular, glial and neuronal cells that comprise this tissue. We propose to now extend the approach developed for this prior work to the specific question of whether similar multi-cell type tissue inflammatory processes underlie opiate withdrawal. We propose a high-dimensional systematic investigation of the CeA inflammatory response in an in vivo rat model of withdrawal aiming to understanding the cell-type specific and molecular framework for neuroinflammatory responses. This will provide potential for improved treatment of opiate withdrawal and its associated pathology. The investigators comprise a collaborative team with the expertise required for this experimental design and analysis, including experts in the molecular effects of opiate exposure and withdrawal in the brain and computational biology and genomics expertise for collection of high dimensional datasets and their analysis and interpretation. We will collect transcriptional and protein data on the molecular and cellular network response in the CeA in order to characterize the cell-type specific contributions of vascular endothelial cells, glia, monocyte lineage cells and neurons to the tissue response. These data will reveal the molecular networks altered within the cell types, their impact on the tissue as a whole, and the resultant neuroinflammatory pathology and neuronal effects that contribute to acute opioid withdrawal's symptomatology.